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Open AccessMethodology A practical approach for the validation of sterility, endotoxin and potency testing of bone marrow mononucleated cells used in cardiac regeneration in compliance

Open Access

Methodology

A practical approach for the validation of sterility, endotoxin

and potency testing of bone marrow mononucleated cells used in

cardiac regeneration in compliance with good manufacturing

practice

Sabrina Soncin, Viviana Lo Cicero, Giuseppe Astori*, Gianni Soldati,

Mauro Gola, Daniel Sürder and Tiziano Moccetti

Address: The Cell Therapy Unit, Cardiocentro Ticino, Via Tesserete 48, CH-6900 Lugano, Switzerland

Email: Sabrina Soncin - sabrina.soncin@cardiocentro.org; Viviana Lo Cicero - viviana.locicero@cardiocentro.org;

Giuseppe Astori* - giuseppe.astori@cardiocentro.org; Gianni Soldati - gianni.soldati@cardiocentro.org; Mauro Gola - mauro.gola@ldm.ch;

Daniel Sürder - daniel.suerder@cardiocentro.org; Tiziano Moccetti - tiziano.moccetti@cardiocentro.org

* Corresponding author

Abstract

Background: Main scope of the EU and FDA regulations is to establish a classification criterion for advanced

therapy medicinal products (ATMP) Regulations require that ATMPs must be prepared under good

manufacturing practice (GMP) We have validated a commercial system for the determination of bacterial

endotoxins in compliance with EU Pharmacopoeia 2.6.14, the sterility testing in compliance with EU

Pharmacopoeia 2.6.1 and a potency assay in an ATMP constituted of mononucleated cells used in cardiac

regeneration

Methods: For the potency assay, cells were placed in the upper part of a modified Boyden chamber containing

Endocult Basal Medium with supplements and transmigrated cells were scored The invasion index was expressed

as the ratio between the numbers of invading cells relative to cell migration through a control insert membrane

For endotoxins, we used a commercially available system based on the kinetic chromogenic LAL-test Validation

of sterility was performed by direct inoculation of TSB and FTM media with the cell product following Eu Ph 2.6.1

guideline

Results and discussion: The calculated MVD and endotoxin limit were 780× and 39 EU/ml respectively The

1:10 and 1:100 dilutions were selected for the validation For sterility, all the FTM cultures were positive after 3

days For TSB cultures, Mycetes and B subtilis were positive after 5 and 3 days respectively The detection limit

was 1-10 colonies

A total of four invasion assay were performed: the calculated invasion index was 28.89 ± 16.82% (mean ± SD)

Conclusion: We have validated a strategy for endotoxin, sterility and potency testing in an ATMP used in cardiac

regeneration Unlike pharmaceutical products, many stem-cell-based products may originate in hospitals where

personnel are unfamiliar with the applicable regulations As new ATMPs are developed, the regulatory framework

is likely to evolve Meanwhile, existing regulations provide an appropriate structure for ensuring the safety and

efficacy of the next generation of ATMPs Personnel must be adequately trained on relevant methods and their

application to stem-cell-based products

Published: 8 September 2009

Journal of Translational Medicine 2009, 7:78 doi:10.1186/1479-5876-7-78

Received: 5 June 2009 Accepted: 8 September 2009

This article is available from: http://www.translational-medicine.com/content/7/1/78

© 2009 Soncin et al; licensee BioMed Central Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The European Union (EU) regulation on advanced

ther-apy medicinal products [1] (ATMP) is entered into force

in all European Member States on December 30, 2008,

and Food and Drug Administration (FDA) recently

prom-ulgated regulations on human cells, tissues, and cellular

and tissue-based products [2] issuing an appropriate

regu-latory structure for the wide range of stem-cell-based

products that may be developed to regenerate damaged

tissues Main scope of the regulations is to establish clear

classification criteria for many new cell-based medicinal

products In particular the European Regulation makes

reference to and is in coherence with the 2004/23/EC

directive on donation, procurement and testing of human

cells and tissues and with directive 2002/98/EC on

human blood and blood components This means that

any use of human cells has to be in compliance with the

quality requirements therein described The European

Regulation is also clear on requiring that all ATMP have to

be prepared according to the good manufacturing practice

(GMP) for medicinal products Stem-cell-based therapies

have existed since the first successful bone marrow

trans-plantations in 1968 [3] Among the ATMPs, bone

mar-row-derived mononuclear cells (BM-MNC), widely used

in cellular therapy protocols, include several populations

of stem cells able to restore vascularization or to

transdif-ferentiate into functional cardiac cells: hematopoietic

stem cells (HSC) which give rise to all mature lineages of

blood [4], mesenchymal stem cells (MSC) and

endothe-lial progenitor cells (EPC) which can be mobilized in the

peripheral blood and give rise to mature endothelial cells

in blood vessels [5] The hematopoietic lineage is

charac-terized by the presence of the CD34 cell-surface antigen

(found in about 1% of human bone marrow

mononucle-ated cells); it has therefore been considered a useful cell

selection target for bone marrow progenitor cells MSC

represent less than 0.1% of the bone marrow cell

popula-tion [6] and are able to generate non hematopoietic

tis-sues including adipocytes, chondrocytes, osteocytes,

myocytes [7,8] and cardiomyocites [9] Angiogenesis and

vascuologenesis are responsible for the development of

the vascular system and are one of the main mechanisms

leading to improved cardiac function after the injection of

BM-MNC [10] Among the CD34+ cells, the CD133

sur-face antigen defines a subset of hematopoietic stem cells

enriched for Endotelial Progenitor Cells (EPCs) [11] The

angiogenic potential of bone marrow cells has been tested

into hind limb ischemia animal models [12] and several

clinical studies are ongoing to evaluate the efficiency of

the intra-arterial administration of BMC into an ischemic

limb [13,14]

During the production of the BM-MNC as medicinal

products, variable amounts of impurities product and

process-related, are introduced into the final product: cells

enter in contact with buffers, reagents and plastics that could be potentially harmful in humans A safety assess-ment of BM-MNC cells prepared using density gradient centrifugation should be done in order to ensure that the finished product do not contain any substance or impu-rity that can have an adverse effect in the patient

BM-MNC should be free from adventitious microbial that could originate from the starting or raw materials or adventitiously introduced during the manufacturing proc-ess In any case, a thorough testing must be performed at the level of finished product in compliance with the meth-odologies described in the EU or United States Pharmaco-poeia (USP), in particular for endotoxin content, sterility and cell potency

Endotoxins are lipo-polysaccharides from gram-negative bacteria and are the most common cause of toxic reactions resulting from contamination with pyrogens: the absence

of bacterial endotoxins in a product implies the absence

of pyrogenic components, provided the presence of non-endotoxin substrates can be ruled out Endotoxins can be detected by using the Limulus amoebocyte lysate (LAL) test; unfortunately, it may be masked by factors interfering with the reaction between the endotoxins and the LAL As

a consequence, the suitability of the regents and materials used and the product itself has to be established The endotoxin limit that can be accepted in a product is based

on the route of administration (intravenous or intrathe-cal), the threshold pyrogenic dose and volume of the injected product Some endotoxin limits have been calcu-lated and can be found in the Pharmacopoeia; for non-compendial items and new drugs, the endotoxin limit should be calculated by the user The Maximum Valid Dilution (MVD) provides an upper bound for dilution that still provides for endotoxin detection at the endo-toxin limit To determine if any interfering characteristics exist, each LAL assay must have a positive product control (PPC) to ensure that endotoxin would be detected if it were present in the sample

Potency is the quantitative measure of biological activity based on the attribute of the product, which is linked to the relevant biological properties The assay demonstrat-ing the biological activity should be based on the intended biological effect which should ideally be related

to the clinical response Basically, two types of potency

assays can be envisioned: in vitro assays using cell systems and in vivo assays using animal models As concerning the

use of bone marrow mononucleated cells in cardiac repair, the importance of characterizing the functionality

of injected cells was recently pointed out [15,16]: to eval-uate the functional activity of the cells obtained after

den-sity gradient centrifugation, authors purposed both in

vitro and in vivo assays Cells were evaluated for

hemat-opoietic colony-forming unit (CFU), and assessment of

mesenchymal stem cells colonies Furthermore, based on

the observation that the migratory capacity of bone

mar-row mononucleated cells predicts the functional

improve-ment after cell transplantation in a hind limb ischemia

model [17] and in humans [18], authors purposed the

assessment of the migration capacity of the cells At the

moment there is no consensus in establishing acceptance

criteria for the migration capacity of BM-MNC in cardiac

regeneration

Cell migration and cell invasion assays measure the ability

of certain cell types to move through a porous membrane

toward a chemoattractant or growth factor In contrast to

cell migration through an open pore, cell invasion

through an occluded pore is dependent on active

enzy-matic degradation of the matrix barrier The Matrigel

Matrix consists of laminin, collagen IV, entactin, and

var-ious growth factors to mimic the basement membrane

Endothelial cells express proteases MMP 2 and 9, which

actively digest the matrix At the end-point of the assay,

invasive cells appear on the underside of the porous

mem-brane and can be quantified

Guidelines for sterility testing of biologics is addressed in

the various worldwide pharmacopeias and in Section 21

of the Code of Federal Regulations (CFR), International

Conference on Harmonisation (ICH) and Food and Drug

Administration Points to Consider documents ATMP

manufactured under GMP conditions require sterility

test-ing performed under GMP guidelines There are two

com-mon types of sterility test methods: the membrane

filtration method that requires the test article to first pass

through a size exclusion membrane capable of retaining

microorganisms and the direct inoculation method

requires the sample to be inoculated directly into test

media For the latter, sample is incubated for 14 days in

the test media It is important to determine if the ATMP

under testing contains elements able to interfere with the

growth of microorganisms within the growth media used

for the assay

Aim of this study is the validation of a commercial system

(Charles River Endosafe PTS) for the determination of

bacterial endotoxins in compliance with Eu

Pharmaco-poeia 2.6.14 (bacterial endotoxins), the validation of the

sterility testing in compliance with eu Pharmacopoeia

2.6.1 (sterility) and the validation of the potency assay in

an ATMP that is constituted of bone-marrow

mononucle-ated cells used in cardiac regeneration

Materials and methods

Testing were performed in the quality control laboratory

of the cell therapy unit of the Cardiocentro Ticino The

Laboratory is authorized and regularly inspected by the Swiss competent authorities

Sample Preparation

For the endotoxin testing and migration assay cells were collected after informed consent from patients enrolled in the "Swiss multicenter intracoronary stem cells study in acute myocardial infarction" (SWISS-AMI, NCT00355186) A total of 50 ml of bone marrow was aspirated into heparin-treated syringes from the posterior iliac crest under local anesthesia Bone marrow was fil-tered by using a 100 μm nylon mesh (BD Falcon TM Cell Strainer, BD Biosciences), diluted 1:1 in Phosphate Buff-ered Saline (PBS), and BM-MNC isolated by density gradi-ent cgradi-entrifugation on Ficoll-PAQUE PREMIUM (General Electric) Cells were washed three times in PBS filtered through a 70 μm nylon mesh (BD Falcon) and then resus-pended in 10 ml of 5% v/v human albumin One ml was collected for migration and invasion assay and endotoxin testing For the sterility testing, peripheral blood mononu-cleated cells were obtained from 50 ml of peripheral blood collected from patients immediately after an acute myocardial infarction (AMI) subjected to standard phar-macological therapy

Cell Characterization

For the immunophenotype, bone marrow and BM-MNC cells were stained in quadruplicate with anti CD45 FITC (Beckman Coulter, USA), anti CD34 PC7 (Becton Dickin-son, San Jose, USA), anti CD133 PE (Miltenyi, Bergisch-Gladbach, DE) and with 7-AAD (Beckman Coulter, USA) for the cell viability test Death cells were excluded from the analysis Analyses were performed using a Cytomics

FC 500 flow cytometer (Beckman Coulter) acquiring at least 100.000 events Isotype-matched murine FITC, PC-7, and PE conjugated immunoglobulins were used as con-trols Cell phenotype was determined by using an ABX Micros 60 (Horiba Diagnostics, France)

Migration and Invasion Assay

A total of 1 × 106 BM-MNC collected from acute myocar-dial infarction patients subjected to standard pharmaco-logical therapy were resuspended in 500 μl of 5% v/v human albumin For the migration assay, cells were placed in the upper part of an 8.0 μm untreated polyeth-ylene terephthalate membrane 24-well cell culture insert (Becton Dickinson, CA) For the invasion assay cells were placed in the upper part of a modified Boyden chamber Matrigel Invasion Camber (BioCoat Matrigel invasion chamber, Becton Dickinson, CA): the chamber consist of

a 24-well Cell Culture insert with an 8 μm pore size PET membrane, uniformly coated with Matrigel Matrix The matrix provides a barrier to non-invasive cells while pre-senting an appropriate protein structure for invading cells

to penetrate before passing through the membrane Both

chambers were then placed in a 24-well culture dish

con-taining 500 μl of Endocult Basal Medium supplemented

with Endocult Single Quots (Stemcells Technologies,

Van-couver, Canada) and 20% fetal calf serum (Figure 1) After

24 hours of incubation at 37°C, 5% v/v CO2

transmi-grated cells were counted Assays were run in duplicates

Endotoxin Testing

Description of the PTS Endosafe system

The Endosafe portable test system is based on the kinetic

chromogenic LAL-test that is based on the cleavage of a

synthetic substrate by an enzyme produced in the reaction

of the lysate in the presence of endotoxin The system

con-sists of LAL reagents and endotoxin controls in the form

of a single-use polystyrene cartridges The cartridges are

potency tested, spike recovery is performed and the

cali-bration code is determined The calicali-bration code contains

the cartridge test parameters that were determined during

potency testing as well as the archived curve for that batch

of cartridges The color intensity developed is

propor-tional to the endotoxin concentration Each cartridge

con-sists of two sample channels and two spiked channels,

consistent with current Pharmacopoeia guidance for

licensed quantitative LAL methods Each reservoir con-tains a specific amount of LAL reagent, synthetic chro-mogenic substrate, control standard endotoxin (CSE) and buffers uniformly embedded in the cartridge The car-tridge is inserted into a dedicated reader and 25 μL of the prepared sample are dispensed into the four reservoirs The reader draws, mixes and incubates the sample with the various reagents at programmed time intervals before transferring it to the optical chambers The portable spec-trophotometer then monitors the change in the optical density and calculates the endotoxin level based on the resulting kinetic values Cartridges with 5-0.050 EU/mL sensitivity were used in this study Results are automati-cally multiplied by the dilution factor entered into the Endosafe system With the correct dilution the unit achieves results in approximately 15 min

Preparation of the inhibition/enhancement test and preparation of the cell therapy product dilution series

The calculated MVD and endotoxin limit for the ATMP were 780× and 39 EU/ml respectively The inhibition/ enhancement test was done by using the Charles River R+D Inhibition/Enhancement cartridges (range 5-0.05 EU/ml ) and by testing the cell product undiluted and

Schematic representation of the invasion assay

Figure 1

Schematic representation of the invasion assay BM-MNC cells were resuspended in 5% v/v human albumin and placed

in the upper part of a modified Boyden chamber Matrigel invasion chamber The chamber consist of a 24-well cell culture insert with an 8 μm pore size PET membrane, uniformly coated with Matrigel matrix The matrix provides a barrier to non-invasive cells while presenting an appropriate protein structure for invading cells to penetrate before passing through the membrane The chamber was then placed in a 24-well culture dish containing 500 μl of Endocult basal medium supplemented with Endoc-ult single quots (Stemcells technologies, Vancouver, Canada) and 20% fetal calf serum After 24 h of incubation transmigrated cells were counted

24 h

Matrigel Matrix occluding the 8.0 μm PET membrane

Chemoattractant

Invading cells

diluted in pyrogen-free water as follows: 1:10; 1:100;

1:500; 1:700; 1:780

This preliminary assay was performed with the aim to find

the dilution where the spiked endotoxin can be detected

without inhibiting or enhancing the test Once prepared,

the cartridge was inserted in the Endosafe PTS and loaded

with 25 μl of the solution in each well Results were scored

after 20 minutes of incubation at 37°C

The ATMP was diluted in LAL reagent water (Charles River

) to 1:10 and 1:100 in pyrogen-free tubes and then loaded

in the system All the tubes, water and pipette-tips were

pyrogen-free certified

Sterility Testing

Sterility testing was carried out under aseptic conditions

regularly monitored by appropriate sampling of the

work-ing area and by carrywork-ing out appropriated controls as

spec-ified in on GMP documents

Growth promotion test (GPT)

Sterility of the culture media Fluid thyoglicollate medium

(FTM) and soya-bean casein digest medium (TSB) used

for the culture of anaerobic and fungi/aerobic bacteria

(THIOC-T and TSB-T, bioMerieux SA, Switzerland) was

performed by incubating two vials of medium for 14 days

at 32.5°C and 22.5°C respectively Growth promotion

test was performed by inoculating FTM media with

10-100 colony-forming units (UFC) of Bacillus subtilis ATCC

6633; Staphylococcus aureus ATCC 6538; Pseudomonas

aeru-ginosa ATCC 9027; Clostridium sporogenes ATCC 19404

and TSB media with 10-100 UFC of Candida albicans

ATCC 10231; Aspergillus niger ATCC 16404 and Bacillus

subtilis ATCC 6633 (all from Quanti-Cult, Remel, Lenexa,

KS) Media were incubated as described for five and three

days respectively Culture plates were inoculated in

paral-lel in order to check the viability of the micro-organisms

Testing was also performed by using the following

bacte-rial strains isolated from bioburden in clean room:

Sphingobacterium multivorum All testing were performed

in duplicate Bacterial identifications were performed by

Gram-staining and by using the mini API detection system

(bioMerieux SA, Switzerland) The ID32 and ATB test

strips were used for the strain identification (bioMerieux

SA, Switzerland)

Validation test

Validation was performed by direct inoculation of TSB

and FTM media with 1% of the total volume of the

prod-uct under validation as stated in European

Pharmaco-poeia (2.6.27) For the latter, 500 μl of whole blood and

100 μl of the BM-MNC were inoculated together with

1-10 UFC and 1-10-1-100 Colony-forming units of the bacterial

strains used in the growth promotion test and incubated

as above described A growth promotion test was per-formed as a positive control If clearly visible growth of micro-organisms is obtained after incubation in presence

of blood and the ATMP, the product possesses no antimi-crobial activity under the conditions of the test, and the sterility may be then carried out without further modifica-tion

Data Analysis

For the endotoxin testing, a test result was considered valid when the percentage of spike recovery was between 50% and 200% with a coefficient of variation less than 25%

For the sterility testing, the detection limit represent the lowest bacterial concentration in the inoculums that the system can evidence The specificity of the system repre-sent its ability to detect the single micro-organism in the inoculums and the detection limit represent the lowest micro-organism number in the sample that the system can detect The robustness of the system represent its abil-ity to obtain identical results when using different prod-ucts, medium from different lots in different working days

For the invasion assay, data were expresses as the percent invasion through the Matrigel matrix and membrane rel-ative to the migration through the 8.0 μm untreated Mem-brane (invasion index) The Assay was considered positive when at least ≥10% of the inoculate cells maintain their invasion capacity

Results

Cell phenotype

Cell phenotype of whole bone marrow and after density gradient separation are reported in Figure 2 (mean ± SD,

n = 4)

Endotoxin testing

Testing was performed on three BM-MNC obtained from different patients in three different days Patient were sub-jected to standard pharmacological treatment for acute myocardial infarction The mononucleated cells concen-tration in the samples were 18.0 × 106/ml; 15.2 × 106/ml and 16.2 × 106/ml respectively (16.5 ± 1.2 × 106 mean ± SD) with a pH of 6.5

Results of the inhibition/enhancement test are reported in Table 1 Based on the obtained results, the 1:10 and 1:100 dilutions were selected for the validation assay An invalid value, based on acceptance criteria, was observed in the first run for the 1:10 dilution The results of the validation assay are reported in Table 2

Sterility testing

Testing was performed on three whole peripheral blood

and the derived mononucleated fractions from different

patients in three different days Patient were subjected to

standard pharmacological treatment for acute myocardial

infarction The white blood cell concentration in the

mononucleated fraction were 13.0 × 106/ml; 12.2 × 106/

ml and 15.2 × 106/ml respectively (13.5 ± 1.6 × 106 mean

± SD) with a pH of 6.5

For the growth promotion test at the end of the

incuba-tion period, clearly visible growth of micro-organisms was

observed and identity confirmed for all bacterial strains

As concerning the strains isolated from bioburden, S

epi-dermidis 1 growth in both TSB and FTM media at both

concentrations whereas M lylae and S multivorum growth

at both concentrations in TSB medium only For the vali-dation test, all the FTM cultures resulted to be positive after 3 days at both the concentration tested For TSB

cul-tures, Mycetes were positive after 5 days and B subtilis after

three The detection limit of the system was then estab-lished in 1-10 colonies At the end of the incubation period, subcultures in agar plates were performed for all the microbial growth: all the identifications confirmed the starting inoculum confirming the robustness of the sys-tem

Migration and invasion assay

A total of four assays were performed in different days For all the samples a significant invasion index was observed: 28.89 ± 16.82% (mean ± SD) Complete results are reported in Figure 3

Discussion

Cellular therapy is an emerging field in medicine; all the stem cell medicinal products must be in compliance with principles and guidelines of good manufacturing practice

Phenotypical analysis of whole bone marrow cells and after density gradient centrifugation (bone marrow selected cells) (n = 4)

Figure 2

Phenotypical analysis of whole bone marrow cells and after density gradient centrifugation (bone marrow selected cells) (n = 4).

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

% VIABILITY % CD34/CD45 % COEXPR

Table 1: Results of the inhibition/enhancement test

in respect of medicinal products and investigational

medicinal products for human use When any new

prepa-ration or method of prepaprepa-ration is adopted, steps should

be taken to demonstrate its suitability for routine

process-ing: the defined process, using the materials and

equip-ment specified, should be validated in order to produce

cells of the required quality

For certain ATMP that must be administered immediately

and that cannot be cryopreserved without damaging the

cell viability and quality, the availability of rapid testing method for endotoxin and sterility testing is fundamental For the latter, traditional methods, including kinetic chro-mogenic, kinetic turbidimetric and gel-clot LAL assay sys-tems, have been widely used in the pharmaceutical industry Unfortunately, all of these methods are time-consuming (several hours) and become problematic if time-sensitive ATMPs products must be immediately released In the present paper, we have demonstrated that

Migration and invasion assay results for bone marrow derived mononucleated cells

Figure 3

Migration and invasion assay results for bone marrow derived mononucleated cells.

0

10

20

30

40

50

60

70

80

SAMPLE

% MIGRATION

% INVASION INVASION INDEX

Table 2: Results of the validation assay

Sample result (EU/mL) <0.532 <.513 <0.500 <0.500 <0.500 <0.500 <0.500 <0.500 <0.500 <0.500 <0.500 <0.500

the PTS endosafe system can be validated for the

endo-toxin testing of BM-MNC in compliance with European

and United States Pharmacopoeia The time required by

the system was approximately 15 min, making it

particu-larly useful as an immediate release testing, where the aim

is to prepare and administer the product within a short

time period

Sterility testing is regulated by USP 21CFR610.12 and by

Eu Pharmacopoeia 2.6.1 We have successfully validated

the sterility testing of a mononucleated cell preparation:

the sensitivity of the system for the ATCC and bioburden

bacterial strains here considered was 1-10 UFC in the

inoculums and cultures were positive after approximately

48 hours of incubation

Recently, a rapid microbiological control strategy for

cel-lular products has been issued in EU and USP

Pharmaco-poeias based on the use of rapid detection systems as the

BacT/Alert 3D (bioMerieux, Durham, USA) or the Bactec

(Becton Dickinson, Franklin Lake, USA) Those systems

are in general non destructive, allowing a faster detection

when compared to TSB/FTM testing, and products can be

released after 7 days Unfortunately, the microbial growth

of certain bacterial strains in those systems is still

contro-versial; as a consequence, those method should be strictly

validated both using the prescribed ATCC strains and by

using bioburden isolates

All biological products must meet prescribed

require-ments of safety, purity and potency and no lot of any

licensed product may be released by the manufacturer

prior to the completion of tests for conformity with

stand-ards applicable to such product, including potency The

current regulations allow for considerable flexibility in

determining the appropriate measurements of potency

that is necessary for product characterization testing;

how-ever, the complexity of an ATMP product can present

sig-nificant challenges in establishing a potency assays

The migration assay of BM-MNC in response to

endothe-lial growth factors, seems to correlate with the beneficial

effects of the cell infusion after myocardial infarction

[15,16]: this assay has been then purposed as a

quantita-tive biological measure for the activity of the product

related to its specific ability to achieve the given result In

particular, has been suggested that the correlation

between the "in vitro" data and the clinical efficacy may

be obtained by analyzing the outcomes from controlled

clinical studies [19,20] In addition to the migration

assay, here we describe the use of the invasion assay as a

potency testing for BM-MNC cells: we purpose to define as

a minimal criteria to establish cell potency in cardiac

regeneration, the obtainment of an invasion index not

less than 10% We are aware that the cell migration and

invasion results "in vitro" should be correlated with the

"in vivo" effect of the cells and this must be addressed both in a suitable animal model and during a controlled clinical trial of acute myocardial infarction

Basic and clinical scientists, as well as scientists working in the biotechnology and pharmaceutical industries, need an increased awareness of the questions that must be answered before a stem-cell-based product can be used clinically Unlike pharmaceutical products, many stem-cell-based products may originate in academic laborato-ries where researchers are unfamiliar with the applicable regulations As new stem-cell-based therapies are devel-oped, the regulatory framework is likely to evolve Mean-while, existing regulations pertaining to biologic products and human cells, tissues, and cellular and tissue-based products provide an appropriate structure for ensuring the safety and efficacy of the next generation of stem cell-based medicinal products As they conduct research on stem cells, scientists should be aware of the relevant regu-lations and their likely application to this products

Competing interests

The authors declare that they have no competing interests

Authors' contributions

GA wrote the manuscript, SS and VLC performed the experiments, DS performed the sample collections as co-investigator of the Swiss Ami clinical Trial, MG performed literature search, GS and TM participated in study design and coordination All the authors read and approved the final manuscript

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